DNA-mediated transfection techniques of NIH 3T3 cells have been used to demonstrate transforming genes in a number of human cancers. Most of these genes are cellular homologs of the viral ras gene which codes for the phosphoprotein p21. The yeast Saccharomyces cerevisiae contains at least two genes that share significant homology with the ras oncogene family. RAS1, an essential gene on chromosome 15, codes for a protein in which the first 171 amino acid residues are 61% homologous with mammalian ras. This research aims to: (1)\isolate conditional lethal mutations of the RAS gene either by in vivo or in vitro mutagenesis with the phenotype of the mutants at the nonpermissive temperature studied. (2)\Isolate revertants of the conditional RAS mutations. In particular, we will screen for cold-sensitive or temperature-sensitive pseudorevertants of the temperature-sensitive or cold-sensitive mutants, respectively. These pseudorevertants may identify genes whose gene products interact with the RAS protein. The phenotype of extragenic pseudorevertants will be determined independently from the RAS mutation. In addition, the pseudorevertants will be genetically mapped and cloned by complementation in order to eventually identify the gene products. (3)\Construct site-specific mutations in the RAS genes using oligonucleotide directed mutagenesis. Many ras transforming genes contain missense mutations that result in amino acid substitutions at residues 12 or 61. These changes are essential and in some cases sufficient to cause the transforming phenotype. The RAS1 gene codes for a protein that contains amino acid residues at position 12 or 61 that are identical to the residues found in the normal nontransforming mammalian ras protein. Site-directed mutations will be made in RAS1 that result in amino acid substitutions at position 12 or 61. These mutations and others will be transferred into yeast for study in vivo. (X)